Apparatus for genetic transformation

• US 5,120,657 A
• Filed: 10/17/1989
• Issued: 06/09/1992
• Est. Priority Date: 12/05/1986
• Status: Expired due to Term

• Current Assignee: PowderJect Vaccines, Inc. (Bayer AG)
• Original Assignee: Agracetus Incorporated
• Inventors: McCabe, Dennis E., Martinell, Brian J., Glaser, Donald A.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/422,921
• Time in Patent Office: 966 Days
• Field of Search: 435/172.1, 435/172.3, 435/173, 435/287, 435/313, 604/68, 604/69, 604/70, 604/72, 604/140, 604/141, 604/146, 935/52, 935/53, 935/85, 128/24 EL, 73/12, 73/167, 239/81
• US Class Current: 435/285.3
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

An apparatus is disclosed for the genetic transformation of organisms by accelerated particle mediated transformation. Foreign genes are introduced into cells by coating on carrier particles which are physically accelerated into the cells by positioning the carrier particles on a carrier sheet which is accelerated by a device which produces a shock wave. The treated cells are recovered, and a portion of them will contain in their genome the foreign gene. The procedure may be used to create genetically engineered organisms of many types.

Claims

1. Apparatus for injecting carrier particles carrying DNA into living cells comprising:

a body including formed therein a spark discharge chamber;

two electrodes extending into the spark discharge chamber and spaced apart from each other so as to define a spark gap, the electrodes constructed so as to be attached to an external source of high voltage discharge;

a carrier sheet placed spaced above the spark discharge chamber in a fashion such that it may be vertically moved, the carrier sheet receiving the carrier particles thereon;

a retaining screen fixed in place above the carrier sheet; and

a target surface held spaced above the retaining screen and constructed and arranged so as to carry the cells so that when a spark discharge generates a shock wave in the discharge chamber, the shock wave will accelerate the carrier sheet into the retaining screen so that the carrier particles are accelerated into the cells on the target surface.

2. Apparatus as claimed in claim 1 wherein the spark gap between the electrodes is between about 1 and 1.5 millimeters.

3. Apparatus as claimed in claim 1 further including a spacer ring sealably attached to the top of the body forming the spark discharge chamber but below the carrier sheet, with at least a partial opening at its top, so as to provide flow communication between the discharge chamber and the carrier sheet.

4. Apparatus as claimed in claim 1 further including a water droplet bridging the spark gap between the electrodes.

5. Apparatus as claimed in claim 4 in which the body further includes an access opening into the discharge chamber and further comprising an access cover removably placed over access opening to the spark discharge chamber so that access can be had to the electrodes to replace the water droplet.

6. Apparatus as claimed in claim 1 further including a containment vessel surrounding the body forming the discharge chamber, the carrier sheet, the retaining screen, and the target surface so that the acceleration of carrier particles may be performed in a partial vacuum.

7. Apparatus as claimed in claim 6 wherein the containment vessel is filled with at least a partial pressure of helium so that the acceleration of carrier particles may be performed in a helium atmosphere.

8. Apparatus as claimed in claim 1 further comprising an external source of high voltage discharge connected to the electrodes with the external source including voltage adjustment means so that the high voltage discharge can be adjusted in voltage between 1 and 15 kilovolts.

9. Apparatus as claimed in claim 8 wherein the external source further includes a capacitor in which the high voltage discharge energy is stored until ready for discharge through the electrodes.

10. An apparatus for the injection of carrier particles carrying genetic material thereon off of a carrier sheet, onto which the particles are layered, and into living cells comprising

a body having a spark discharge chamber defined therein;

a pair of spaced electrodes extending into the spark discharge chamber;

a source of a discharge of electrical energy switchably connected to the electrodes so as to be able to initiate a spark discharge between the electrodes;

a planar carrier sheet;

the body forming the spark discharge chamber provided with an opening to receive the carrier sheet thereon, the spark discharge chamber being substantially sealed when the carrier sheet is in place on the opening;

a retaining screen for retaining the carrier sheet after it has been accelerated off the spark discharge chamber while permitting the carrier particles to pass through;

a target in the line of travel of the carrier sheet into which the carrier particles will travel after the carrier sheet is restrained on the retaining screen; and

a containment vessel surrounding the spark discharge chamber, the retaining means and the target and isolating them from the atmosphere;

the containment vessel having the atmosphere therein at least partially replaced by helium.

11. The apparatus as claimed in claim 10 wherein the spark discharge chamber is sealed by the carrier sheet being simply placed atop the opening in the spark discharge chamber.

12. An apparatus for the injection of carrier particles carrying genetic material into living cells comprising:

a thin planar carrier sheet;

a plurality of small biologically inert carrier particles coated with genetic material disposed on the surface of the carrier sheet;

a target surface having living cells;

a body forming a discharge chamber having an opening therein, the carrier sheet placed on the opening to seal the opening;

means to generate a shock wave in the discharge chamber sufficient to accelerate the carrier sheet with the carrier particles thereon to travel toward the target surface; and

a retaining screen disposed in the path of travel of the carrier sheet between the discharge chamber and the target surface, the carrier particles then leaving the carrier sheet to travel on toward the target surface.

13. An apparatus as claimed in claim 12 wherein the means for generating the shock wave is adjustable.

14. An apparatus for the injection of carrier particles carrying genetic material into a target including living cells comprising:

a thin planar carrier sheet;

a plurality of small biologically inert carrier particles coated with genetic material disposed on the surface of the carrier sheet;

a body forming a discharge chamber having an opening therein, the carrier sheet placed on the opening to seal the opening;

means for generating a shock wave in the discharge chamber sufficient to accelerate the carrier sheet with the carrier particles thereon to travel toward the target; and

a retaining screen disposed in the path of travel of the carrier sheet between the discharge chamber and the target and positioned and constructed so as to restrain the carrier sheet when the carrier sheet is accelerated at the target, the carrier particles then leaving the carrier sheet to travel on toward the target.

15. An apparatus as claimed in claim 14 further comprising means for substantially replacing atmospheric gases between the carrier sheet and the retaining screen with helium.